Project Summary/Abstract The increasing appreciation of RNA's structure-function relationship has led to a demand for new technologies that enable targeting of specific RNA structures. Such technologies are essential for the development of probes to study RNA function and therapeutics to treat RNA-mediated diseases. However, outside of antibiotics binding the ribosome, structure-specific RNA-binding reagents are very rare. Thus, developing of new technologies that enable structure-specific targeting of RNA remains an important challenge in many fields. The central vision of my research program is to address the deficit of structure-specific RNA-binding reagents using a radically different type of nucleic acid affinity reagent: L-aptamers. L-Aptamers are unique because they are comprised of L-(deoxy)ribose-based nucleic acids (L-DNA and L-RNA), which are mirror images (enantiomers) of natural D-nucleotides. Because oligonucleotides of opposite stereochemistry (D versus L) are incapable of forming contiguous Watson-Crick base pairs with each other, we are able to evolve L-aptamers that adaptively bind structured D-RNA targets through tertiary interactions (shape) rather than primary sequence. In other words, L-aptamers escape the tyranny of Watson-Crick base pairing, enabling a more nuanced mode of molecular recognition to be discovered. As a result, L-aptamers bind structured RNAs with greater affinity and specificity compared to conventional affinity reagent. Binding RNAs based on their shape rather than Watson-Crick base pairing represents a significant departure from traditional oligonucleotide-based approaches and represents a major advance in aptamer technology. During the next five year, my research group aims to further develop L-aptamer technology in order to realize its promise as a practical research and therapeutic tool. In particular, we will focus on incorporation of modified nucleotides that bestow protein-like functionality on L-aptamers, thus generating a novel class of RNA-targeted antibody mimetics. Because these technological developments will be carried out in the context of disease associated RNAs, such as oncogenic microRNAs and viral RNAs, this work will have an immediate impact by generating lead reagents to probe the etiology of disease and develop new therapeutic strategies. In line with my vision, we aim to determine the structure of an L-aptamer?D-RNA complex, which will provide insight into this novel mode of recognition and inform future L-aptamer design.